Biophysical Marker Research Articles

The upregulation of K+ and HCN channels in developing spiral ganglion neurons is mediated by cochlear inner hair cells.

Spiral ganglion neurons (SGNs) are primary sensory afferent neurons that relay acoustic information from the cochlear inner hair cells (IHCs) to the brainstem. The response properties of different SGNs diverge to represent a wide range of sound intensities in an action-potential code. This biophysical heterogeneity is established during pre-hearing stages of development, a time when IHCs fire spontaneous Ca2+ action potentials that drive glutamate release from their ribbon synapses onto the SGN terminals. The role of spontaneous IHC activity in the refinement of SGN characteristics is still largely unknown. Using pre-hearing otoferlin knockout mice (Otof-/-), in which Ca2+-dependent exocytosis in IHCs is abolished, we found that developing SGNs fail to upregulate low-voltage-activated K+-channels and hyperpolarisation-activated cyclic-nucleotide-gated channels. This delayed maturation resulted in hyperexcitable SGNs with immature firing characteristics. We have also shown that SGNs that synapse with the pillar side of the IHCs selectively express a resurgent K+ current, highlighting a novel biophysical marker for these neurons. RNA-sequencing showed that several K+ channels are downregulated in Otof-/- mice, further supporting the electrophysiological recordings. Our data demonstrate that spontaneous Ca2+-dependent activity in pre-hearing IHCs regulates some of the key biophysical and molecular features of the developing SGNs. KEY POINTS: Ca2+-dependent exocytosis in inner hair cells (IHCs) is otoferlin-dependent as early as postnatal day 1. A lack of otoferlin in IHCs affects potassium channel expression in SGNs. The absence of otoferlin is associated with SGN hyperexcitability. We propose that type I spiral ganglion neuron functional maturation depends on IHC exocytosis.

Electro-antibacterial therapy (EAT) to enhance intracellular bacteria clearance in pancreatic cancer cells

Intratumoral bacteria have been implicated in driving tumor progression, yet effective treatments to modulate the tumor microbiome remain limited. In this study, we investigate the use of electroporation in combination with metronidazole to enhance the clearance of intracellular Fusobacterium nucleatum within pancreatic cancer cells. We explore various parameters, including electric field strength, pulse width, and pulse number to assess the permeability of pancreatic cancer cells infected with F. nucleatum, compared to non-infected cells of the same type. We subsequently quantify the clearance of intracellular bacteria when these pulsing schemes are applied to a suspension of infected pancreatic cancer cells in the presence of metronidazole. Our results reveal distinct differences in cell permeability between infected and non-infected cells, identifying a unique biophysical marker for host cells infected with F. nucleatum. We demonstrate that the combinatorial use of electroporation and metronidazole significantly enhances the delivery of metronidazole into host cells, leading to more effective clearance of intracellular F. nucleatum compared to independent treatments; we term this novel approach Electro-Antibacterial Therapy (EAT). EAT holds promise as an innovative strategy for addressing intratumoral bacteria in pancreatic cancer, other malignancies, and potentially treatment-resistant infections, offering new avenues for therapeutic intervention.

Fiber alignment in 3D collagen networks as a biophysical marker for cell contractility

Cells cultured in 3D fibrous biopolymer matrices exert traction forces on their environment that induce deformations and remodeling of the fiber network. By measuring these deformations, the traction forces can be reconstructed if the mechanical properties of the matrix and the force-free matrix configuration are known. These requirements limit the applicability of traction force reconstruction in practice. In this study, we test whether force-induced matrix remodeling can instead be used as a proxy for cellular traction forces. We measure the traction forces of hepatic stellate cells and different glioblastoma cell lines and quantify matrix remodeling by measuring the fiber orientation and fiber density around these cells. In agreement with simulated fiber networks, we demonstrate that changes in local fiber orientation and density are directly related to cell forces. By resolving Rho-kinase (ROCK) inhibitor-induced changes of traction forces, fiber alignment, and fiber density in hepatic stellate cells, we show that the method is suitable for drug screening assays. We conclude that differences in local fiber orientation and density, which are easily measurable, can be used as a qualitative proxy for changes in traction forces. The method is available as an open-source Python package with a graphical user interface.

Stromal cells regulate mechanics of tumour spheroid

The remarkable contractility and force generation ability exhibited by cancer cells empower them to overcome the resistance and steric hindrance presented by a three-dimensional, interconnected matrix. Cancer cells disseminate by actively remodelling and deforming their extracellular matrix (ECM). The process of tumour growth and its ECM remodelling have been extensively studied, but the effect of the cellular tumour microenvironment (TME) has been ignored in most studies that investigated tumour-cell-mediated ECM deformations and realignment. This study reports the integration of stromal cells in spheroid contractility assays that impacts the ECM remodelling and invasion abilities of cancer spheroids. To investigate this, we developed a novel multilayer in vitro assay that incorporates stromal cells and quantifies the contractile deformations that tumour spheroids exert on the ECM. We observed a negative correlation between the spheroid invasion potential and the levels of collagen deformation. The presence of stromal cells significantly increased cancer cell invasiveness and altered the cancer cells' ability to deform and realign collagen gel, due to upregulation of proinflammatory cytokines. Interestingly, this was observed consistently in both metastatic and non-metastatic cancer cells. Our findings contribute to a better understanding of the vital role played by the cellular TME in regulating the invasive outgrowth of cancer cells and underscore the potential of utilising matrix deformation measurements as a biophysical marker for evaluating invasiveness and informing targeted therapeutic opportunities.

μDACS platform: A hybrid microfluidic platform using magnetic levitation technique and integrating magnetic, gravitational, and drag forces for density-based rare cancer cell sorting

Circulating tumor cells (CTCs) are crucial indicators of cancer metastasis. However, their rarity in the bloodstream and the heterogeneity of their surface biomarkers present challenges for their isolation. Here, we developed a hybrid microfluidic platform (microfluidic-based density-associated cell sorting (µDACS) platform) that utilizes density as a biophysical marker to sort cancer cells from the population of white blood cells (WBCs). The platform utilizes the magnetic levitation technique on a microfluidic chip to sort cells based on their specific density ranges, operating under a continuous flow condition. By harnessing magnetic, gravitational, and drag forces, the platform efficiently separates cells. This approach involves a microfluidic chip equipped with a microseparator, which directs cells into top and bottom outlets depending on their levitation heights, which are inversely proportional to their densities. Hence, low-density cancer cells are collected from the top outlet, while high-density WBCs are collected from the bottom outlet. We optimized the sorting efficiency by varying the flow rates, and concentrations of the sorting medium's paramagnetic properties using standard densities of polymeric microspheres. To demonstrate the platform's applicability, we performed hybrid microfluidic sorting on MDA-MB-231 human breast cancer cells and U-937 human monocytes. The results showed efficient sorting of rare cancer cells (≥100 cells/mL) from serum samples, achieving a sorting efficiency of ∼70% at a fast-processing speed of 1 mL h−1. This label-free approach holds promise for rapid and cost-effective CTC sorting, facilitating in-vitro diagnosis and prognosis of cancer.

Modelling the cost-effectiveness of subepidermal moisture measurement as part of a process of assessment and intervention to prevent hospital-acquired pressure ulcers.

Skin tissue assessment is traditionally used to identify early signs of pressure damage from changes observed at the skin surface. However, the early onset of tissue damage induced by pressure and shear forces is likely to be on soft tissues beneath the surface of the skin. Subepidermal moisture (SEM) is a biophysical marker for the detection of early and deep pressure-induced tissue damage. Measurement of SEM can detect early pressure ulcers up to 5 days before visible skin changes occur. The aim of this study was to evaluate the cost-effectiveness of SEM measurement compared with visual skin assessment (VSA). A decision-tree model was developed. Outcomes are the incidence of hospital-acquired pressure ulcers, quality-adjusted life-years (QALYs) and costs to the UK National Health Service. Costs are at 2020/21 prices. The effects of parameter uncertainty are tested in univariate and probabilistic sensitivity analysis. In a representative NHS acute hospital, the incremental cost of SEM assessment as an adjunct to VSA is -£8.99 per admission, and SEM assessment is expected to reduce the incidence of hospital-acquired pressure ulcers by 21.1%, reduce NHS costs and lead to a gain of 3.634 QALYs. The probability of cost-effectiveness at a threshold of £30 000 per quality-adjusted life year is 61.84%. Pathways that include SEM assessment make it possible to implement early and anatomy-specific interventions which have the potential to improve the effectiveness of pressure ulcer prevention and reduce healthcare costs.

Sputum handling for rheology

The rheology of sputum is viewed as a powerful emerging biophysical marker for monitoring muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and non-CF bronchiectasis (NCFB). However, there is no unified practice to process sputa from collection to analysis, which can lead to highly variable, and sometimes inconsistent results. The main objective of this study is to bring light into the handling of sputum samples to establish a standardised and robust protocol before rheological measurements. Sputum collected from 22 CF and 10 NCFB adults, was divided into control (vortexed and fresh: non-heated and non-frozen) and three treated conditions (either non-vortexed, heated or frozen). In addition, 6 CF expectorations were used to study the dynamics of ageing over 24 h. Sputum’s mechanical properties were measured with a rotational rheometer to obtain their properties at rest, elastic (G') and viscous moduli (G''), and at the onset of flow, critical deformation (gamma _c) and critical stress (sigma _c). We demonstrate that heating sputum is completely destructive while freezing sputa at -80,,^{circ }hbox {C} has no discernible effect on their rheology. We also show that the variability of rheological measurements largely resulted from the sample’s macroscopic heterogeneity, and can be greatly reduced by non-destructive vortex homogenisation. Finally, we observed contrasted ageing effects as a fonction of purulence: while the viscoelasticity of purulent samples reduced by half within 6 h after collection, semi-purulent samples did not evolve. These results guide towards a robust unified protocol for simple sputum handling in rheometry. We therefore suggest to vortex and snap freeze sputum samples immediately after collection when direct testing is not possible.

Recent advances in deformation-assisted microfluidic cell sorting technologies.

Cell sorting is an essential prerequisite for cell research and has great value in life science and clinical studies. Among the many microfluidic cell sorting technologies, label-free methods based on the size of different cell types have been widely studied. However, the heterogeneity in size for cells of the same type and the inevitable size overlap between different types of cells would result in performance degradation in size-based sorting. To tackle such challenges, deformation-assisted technologies are receiving more attention recently. Cell deformability is an inherent biophysical marker of cells that reflects the changes in their internal structures and physiological states. It provides additional dimensional information for cell sorting besides size. Therefore, in this review, we summarize the recent advances in deformation-assisted microfluidic cell sorting technologies. According to how the deformability is characterized and the form in which the force acts, the technologies can be divided into two categories: (1) the indirect category including transit-time-based and image-based methods, and (2) the direct category including microstructure-based and hydrodynamics-based methods. Finally, the separation performance and the application scenarios of each method, the existing challenges and future outlook are discussed. Deformation-assisted microfluidic cell sorting technologies are expected to realize greater potential in the label-free analysis of cells.

Personalized stratification of pregnancy care for small for gestational age neonates from biophysical markers at midgestation

Antenatal identification of pregnancies at high risk of delivering small for gestational age neonates may improve the management of the condition and reduce the associated adverse perinatal outcomes. In a series of publications, we have developed a new competing-risks model for small for gestational age prediction, and we demonstrated that the new approach has a superior performance to that of the traditional methods. The next step in shaping the appropriate management of small for gestational age is the timely assessment of these high-risk pregnancies according to an antenatal stratification plan. This study aimed to demonstrate the stratification of pregnancy care based on individual patient risk derived from the application of the competing-risks model for small for gestational age that combines maternal factors with sonographic estimated fetal weight and uterine artery pulsatility index at midgestation. This was a prospective observational study of 96,678 singleton pregnancies undergoing routine ultrasound examination at 19 to 24 weeks of gestation, which included recording of estimated fetal weight and measurement of uterine artery pulsatility index. The competing-risks model for small for gestational age was used to create a patient-specific stratification curve capable to define a specific timing for a repeated ultrasound examination after 24 weeks. We examined different stratification plans with the intention of detecting approximately 80%, 85%, 90%, and 95% of small for gestational age neonates with birthweight <3rd and <10th percentiles at any gestational age at delivery until 36 weeks; all pregnancies would be offered a routine ultrasound examination at 36 weeks. The stratification of pregnancy care for small for gestational age can be based on a patient-specific stratification curve. Factors from maternal history, low estimated fetal weight, and increased uterine artery pulsatility index shift the personalized risk curve toward higher risks. The degree of shifting defines the timing for assessment for each pregnancy. If the objective of our antenatal plan was to detect 80%, 85%, 90%, and 95% of small for gestational age neonates at any gestational age at delivery until 36 weeks, the median (range) proportions (percentages) of population examined per week would be 3.15 (1.9-3.7), 3.85 (2.7-4.5), 4.75 (4.0-5.4), and 6.45 (3.7-8.0) for small for gestational age <3rd percentile and 3.8 (2.5-4.6), 4.6 (3.6-5.4), 5.7 (3.8-6.4), and 7.35 (3.3-9.8) for small for gestational age <10th percentile, respectively. The competing-risks model provides an effective personalized continuous stratification of pregnancy care for small for gestational age which is based on individual characteristics and biophysical marker levels recorded at the midgestation scan.

A STUDY TO CORRELATE SERUM ANTI MULLERIAN HORMONE, BASAL FOLLICLE STIMULATING HORMONE AND ANTRAL FOLLICLE COUNT IN PRIMARY INFERTILITY AS A MEASURE OF OVARIAN RESERVE

Objectives: This study aims to find the correlation between anti mullerian hormone (AMH) and follicle stimulating hormone (FSH) and to observe the above mentioned hormones relation with antral follicle count (AFC) in patients with primary infertility. Methodology: This is a cross-sectional correlation study in which 60 patients with primary infertility meeting inclusion criteria, attending infertility clinic in Chalmeda Ananda Rao Institute Of Medical Sciences, Karimnagar, between October 2020and April 2022 were enrolled by simple random sampling. Detailed menstrual, obstetric, coital and medical history was obtained. On the third day of the spontaneous cycle, all patients were investigated with a transvaginal scan to assess the number of antral follicles and a fasting venous blood sample was obtained for the measurement of serum AMH and serum basal FSH level. Results: Basal serum FSH shows a moderately strong negative correlation with antral follicle count (AFC) (r=0.65 p=&lt;0.001) and a strong negative correlation with anti mullerian hormone (AMH) (r=0.69 and p=&lt;0.001). However, the strongest correlation between a biochemical marker and biophysical marker of ovarian reserve is between anti mullerian hormone (AMH) and antral follicle count (AFC) with a very strong positive correlation with a correlation co-efficient r=0.89 (p=&lt;0.001). Conclusion: Serum AMH best correlates with the antral follicle count. Antral follicle counts although an efficient test to detect ovarian reserve is uncomfortable for the patient as it has to be done during menstrual flow. Serum AMH with minimal intracycle and intercycle variation is a more convenient marker to assess ovarian reserve while it maintains the accuracy of AFC.

Biophysical Markers of Suspected Preeclampsia, Fetal Growth Restriction and The Two Combined—How Accurate They Are?

Objectives—To conduct a secondary analysis of prediction accuracy of biophysical markers for suspected Preeclampsia (PE), Fetal Growth Restriction (FGR) and the two combined near delivery in a Slovenian cohort. Methods—This was a secondary analysis of a database of a total 125 Slovenian pregnant women attending a high-risk pregnancy clinic due to suspected PE (n = 31), FGR (n = 16) and PE + FGR (n = 42) from 28–39 weeks gestation and their corresponding term (n = 21) and preterm (PTD, n = 15) controls. Data for Mean Arterial blood Pressure (MAP) and Uterine artery pulsatility index (UtA PI) estimated by Doppler sonography were extracted from the database of patients who were tested at admission to the high-risk clinic with the suspected complications. The reactive hyperemia index (RHI), and the Augmentation Index (AIX%) were extracted from the patient database using measured values obtained with the assistance of the Endo PAT, a device set to measure the signal of the peripheral arterial tone (PAT) from the blood vessels endothelium. Linear regression coefficients, Box and Whisker plots, Area under the Curve (AUC) of receiver Operation Characteristic (ROC) curves, and multiple regression were used to assess the marker accuracy using detection rate (DR) and false-positive rate (FPR) and previously reported cut-offs for estimating the positive and negative predictive value (NPV and PPV). The SPSS non-parametric statistics (Kruskal Wallis and Mann–Whitney) and Spearman’s regression coefficient were used to assess marker accuracy; p &lt; 0.05 was considered significant. Results—MAP values reached diagnostic accuracy (AUC = 1.00, DR = 100%) for early PE cases delivered &lt; 34, whereas UtA Doppler PI values yielded such results for early FGR &lt; 34 weeks and the two combined reached such accuracy for PE + FGR. To reach diagnostic accuracy for all cases of the complications, the Endo PAT markers with values for MAP and UtA Doppler PI were required for cases near delivery. Multiple regression analyses showed added value for advanced maternal age and gestational week in risk assessment for all cases of PE, FGR, and PE + FGR. Spearman’s regression coefficient yielded r &gt; 0.6 for UtA Doppler PI over GA for PE and FGR, whereas for RHI over BMI, the regression coefficient was r &gt; 0.5 (p &lt; 0.001 for each). Very high correlations were also found between UtA Doppler PI and sFlt-1/PlGF or PlGF (r = −0.495, p &lt; 0.001), especially in cases of FGR. Conclusion—The classical biophysical markers MAP and UtA Doppler PI provided diagnostic accuracy for PE and FGR &lt; 34 wks gestation. A multiple biophysical marker analysis was required to reach diagnostic accuracy for all cases of these complications. The UtA Doppler PI and maternal serum sFlt-1/PlGF or PlGF were equally accurate for early cases to enable the choice of the markers for the clinical use according to the more accessible method.

Значение симпатовагального баланса как биофизического маркера преэклампсии

Преэклампсия (ПЭ) является одним из серьезных осложнений беременности, которое приводит к синдрому полиорганной недостаточности. Целью исследования было изучение взаимосвязи симпатовагального баланса и типа центральной материнской гемодинамики у беременных с ПЭ. Материалы и методы. Было проведено изучение типа центральной материнской гемодинамики и вариабельности сердечного ритма у 102 пациенток в 32–37 недель беременности. 30 из них были с физиологическим течением процесса гестации и составили группу I (контроля). Во II группе под наблюдением находилось 40 беременных с ПЭ легкой и средней степени. К III группе были отнесены 32 пациентки с ПЭ тяжелой степени. Результаты. Показатели вариабельности сердечного ритма матери отражали гиперсимпатикотонию на фоне ПЭ. Полученные данные подтверждали известную гипердинамическую модель легкой и среднетяжелой ПЭ. Сохранность гестационной гиперволемии на фоне повышенного периферического сопротивления сосудов приводила к увеличению сердечного выброса. Переход к гиповолемии и низкому сердечному выбросу отражал гиподинамическую гемоциркуляцию на фоне тяжелой ПЭ. Повышение симпатовагального баланса было признаком вазоконстрикции и гипоперфузии. Это было подтверждено наличием сильной корреляции между симпатовагальным балансом и общим периферическим сопротивлением сосудов (R = 0,70; р &lt; 0,05), симпатовагальным балансом и сердечным индексом (R = –0,63; р &lt; 0,05 ). Вывод. Повышение симпатовагального баланса более 2,0 было биофизическим маркером ПЭ.

A machine learning algorithm for early detection of heel deep tissue injuries based on a daily history of sub-epidermal moisture measurements.

Sub‐epidermal moisture is an established biophysical marker of pressure ulcer formation based on biocapacitance changes in affected soft tissues, which has been shown to facilitate early detection of these injuries. Artificial intelligence shows great promise in wound prevention and care, including in automated analyses of quantitative measures of tissue health such as sub‐epidermal moisture readings acquired over time for effective, patient‐specific, and anatomical‐site‐specific pressure ulcer prophylaxis. Here, we developed a novel machine learning algorithm for early detection of heel deep tissue injuries, which was trained using a database comprising six consecutive daily sub‐epidermal moisture measurements recorded from 173 patients in acute and post‐acute care settings. This algorithm was able to achieve strong predictive power in forecasting heel deep tissue injury events the next day, with sensitivity and specificity of 77% and 80%, respectively, revealing the clinical potential of artificial intelligence‐powered technology for hospital‐acquired pressure ulcer prevention. The current work forms the scientific basis for clinical implementation of machine learning algorithms that provide effective, early, and anatomy‐specific preventive interventions to minimise the occurrence of hospital‐acquired pressure ulcers based on routine tissue health status measurements.

Degradation of Red Blood Cell Deformability during Cold Storage in Blood Bags

RBC transfusions are a life-saving procedure, aiding both chronic and acute patients in restoring tissue oxygenation. The ability to store collected RBC units for prolonged periods has been one of the most transformative advances in medicine, significantly improving the reliability and the speed of access to blood. However, RBCs undergo a number of metabolic, structural, and biochemical changes during storage, collectively known as the storage lesion, that is detrimental to the quality of the RBC. A major challenge is the ability to evaluate the extent of the storage lesion, and thus the quality of the stored RBC unit directly prior to transfusion.The storage lesion can directly or indirectly reduce the ability of the RBC to deform through the small openings in the microvasculature. Rigid RBCs pose a risk of sequestration in capillaries, impeding blood flow and reducing tissue oxygenation, and are more likely to be cleared out by endothelial macrophages. Studies have shown that there is a loss in RBC deformability during storage and that the rate of RBC deformability loss is donor-dependent. Thus, RBC deformability can be a valuable and reliable biophysical marker of RBC unit quality.Currently, there is a need for a reliable measurement technique that is repeatable and sensitive enough to observe individual differences in RBC deformability in healthy donors, to enable quality control testing of RBC units. We have developed the microfluidic ratchet device, which sorts RBCs based on their deformability, allowing the measurement of both rigid and deformable sup-populations of RBCs within the sample, and generating a unique deformability curve. Here, we use this assay to predict the quality of stored RBC units.We assessed the deformability of 14 healthy donor RBC units through 8 weeks of cold storage at 4°C, which is 2 weeks beyond the Canadian Blood Services approved 6-week standard in Canada. We measured RBC deformability, standard hematological parameters (MCV, MCHC, MCH, and RDW), and hemolysis levels at the time of RBC unit manufacture (week 0), followed by weeks 2, 4, 6, and 8. The microfluidic ratchet device operates by forcing RBCs to deform and travel through rows of tapered constrictions. Constriction size changes from 7.5 to 1.5 µm and is reflective of the microvasculature and vessel opening sizes encountered by RBCs in circulation. RBCs are sorted into 12 distinct outlets based on their deformability. Distribution of RBCs in outlets 1-12 can be quantified and used to calculate the cumulative distribution curve. The cumulative distribution curve provides a distinct deformability signature of each individual RBC sample, which can be defined as rigidity score (RS). RS provides an easy metric to compare the changes in RBC deformability throughout storage (ΔRS) in a single donor as well as across multiple donors.We show that there are both donor- and sex-specific differences in the RBC deformability signatures of stored RBC units. We observed significant inter-donor variability in RBC deformability measured on the day of the RBC unit manufacture, where male donors showed a more stable RBC deformability range (n=8, RS=3.00±0.18) compared to female donors (n=6, RS= 3.29±0.48). The average RS scores were stable between weeks 0-2 (ΔRS 0.07) and showed a reduction in deformability between weeks 1-6 (ΔRS 0.35), with the greatest loss seen between weeks 6-8 (ΔRS 0.42) of cold storage. Interestingly, the response to cold storage is variable, with ΔRS 0.22 to 0.90, suggesting that some donors are more susceptible to storage related changes in RBC deformability than others. Notably, the change in RS over time was donor-specific and did not correlate with RBC deformability at week 0. The majority of RBCs from male donors (ΔRS 0.485, p<0.05), but none of the female donors (ΔRS 0.172) showed changes in deformability during cold storage, suggesting that RBCs from female donors degrade at a slower rate compared to RBCs from male donors.The ability to profile RBC deformability at the individual blood bag level may help identify more stable RBCs for use in chronic and sensitive patients, or RBC units that can be safely stored beyond the 6-week storage window. DisclosuresNo relevant conflicts of interest to declare.

A pre-screening strategy to assess resected tumor margins by imaging cytoplasmic viscosity and hypoxia.

To assure complete tumor removal, frozen section analysis is the most common procedure for intraoperative pathological assessment of resected tumor margins. However, during one operation, multiple biopsies may be sent for examination, but only few of them are made into cryosections because of the complex preparation protocols and time-consuming pathological analysis, which potentially increases the risk of overlooking tumor involvement. Here, we propose a fluorescence-based pre-screening strategy that allows high-throughput, convenient, and fast gross assessment of resected tumor margins. A dual-activatable cationic fluorescent molecular rotor was developed to specifically illuminate live tumor cells' cytoplasm by emitting two different fluorescence signals in response to elevations in hypoxia-induced nitroreductase (a biochemical marker) and cytoplasmic viscosity (a biophysical marker), two characteristics of cancer cells. The ability of the fluorescent molecular rotor in detecting tumor cells was evaluated in mouse and human specimens of multiple tissues by comparing with hematoxylin and eosin staining. Importantly, the fluorescent molecular rotor achieved 100 % specificity in discriminating lung and liver cancers from normal tissue, allowing pre-screening of the tumor-free surgical margins and promoting clinical decision. Altogether, this type of fluorescent molecular rotor and the proposed strategy may serve as a new option to facilitate intraoperative assessment of resected tumor margins.

High-Throughput Characterization of Cell Adhesion Strength Using Long-Channel Constriction-Based Microfluidics.

The adhesion strength of a cancer cell is a valuable biophysical marker of its metastatic potential, tightly associated with various metastatic processes; for example, cancer cells escape from a primary tumor, and circulating tumor cells (CTCs) are anchored to the vessel wall. Although constriction-based microfluidics can realize the high-throughput characterization of single-cell deformability, due to the influence of cell size heterogeneity, accurately evaluating the adhesion strength of a cancer cell at high throughputs in constriction remains difficult. In this paper, we first proposed an approach for the assessment of adhesion strength of BGC-823 and SGC-7901 cell lines at high throughputs based on a friction coefficient using the constant velocity stage of cell transit in a long-channel constriction. Cell size was proven to be independent of adhesion strength by cell detachment assay; however, it has large effects on cell transit velocity in constriction. Therefore, the linear elasticity of a completely deformed cell in constriction is simplified as a compressed spring model, effectively reducing the influence of cell size heterogeneity. Theoretically, our proposed model can well offset the influence of cell size by cell transit velocity, while our experimental results indicate that the friction coefficient has a good linear relationship with the logarithm of the adhesion strength too. Therefore, our proposed approach can realize accurate characterization of cell adhesion strength at high throughputs using long-channel constriction-based microfluidics. Hence, this work might enrich the functions of constriction-based microfluidics and bring new insights into the characterization of mechanical phenotypes.

Contacts with Macrophages Promote an Aggressive Nanomechanical Phenotype of Circulating Tumor Cells in Prostate Cancer.

Aggressive tumors of epithelial origin shed cells that intravasate and become circulating tumor cells (CTC). The CTCs that are able to survive the stresses encountered in the bloodstream can then seed metastases. We demonstrated previously that CTCs isolated from the blood of prostate cancer patients display specific nanomechanical phenotypes characteristic of cell endurance and invasiveness and patient sensitivity to androgen deprivation therapy. Here we report that patient-isolated CTCs are nanomechanically distinct from cells randomly shed from the tumor, with high adhesion as the most distinguishing biophysical marker. CTCs uniquely coisolated with macrophage-like cells bearing the markers of tumor-associated macrophages (TAM). The presence of these immune cells was indicative of a survival-promoting phenotype of "mechanical fitness" in CTCs based on high softness and high adhesion as determined by atomic force microscopy. Correlations between enumeration of macrophages and mechanical fitness of CTCs were strong in patients before the start of hormonal therapy. Single-cell proteomic analysis and nanomechanical phenotyping of tumor cell-macrophage cocultures revealed that macrophages promoted epithelial-mesenchymal plasticity in prostate cancer cells, manifesting in their mechanical fitness. The resulting softness and adhesiveness of the mechanically fit CTCs confer resistance to shear stress and enable protective cell clustering. These findings suggest that selected tumor cells are coached by TAMs and accompanied by them to acquire intermediate epithelial/mesenchymal status, thereby facilitating survival during the critical early stage leading to metastasis. SIGNIFICANCE: The interaction between macrophages and circulating tumor cells increases the capacity of tumor cells to initiate metastasis and may constitute a new set of blood-based targets for pharmacologic intervention.

Biological Applications of Electron Paramagnetic Resonance Viscometry Using a 13C-Labeled Trityl Spin Probe.

Alterations in viscosity of biological fluids and tissues play an important role in health and diseases. It has been demonstrated that the electron paramagnetic resonance (EPR) spectrum of a 13C-labeled trityl spin probe (13C-dFT) is highly sensitive to the local viscosity of its microenvironment. In the present study, we demonstrate that X-band (9.5 GHz) EPR viscometry using 13C-dFT provides a simple tool to accurately measure the microviscosity of human blood in microliter volumes obtained from healthy volunteers. An application of low-field L-band (1.2 GHz) EPR with a penetration depth of 1–2 cm allowed for microviscosity measurements using 13C-dFT in the living tissues from isolated organs and in vivo in anesthetized mice. In summary, this study demonstrates that EPR viscometry using a 13C-dFT probe can be used to noninvasively and rapidly measure the microviscosity of blood and interstitial fluids in living tissues and potentially to evaluate this biophysical marker of microenvironment under various physiological and pathological conditions in preclinical and clinical settings.

Prediction of preeclampsia throughout gestation with maternal characteristics and biophysical and biochemical markers: a longitudinal study

The current approach to predict preeclampsia combines maternal risk factors and evidence from biophysical markers (mean arterial pressure, Doppler velocimetry of the uterine arteries) and maternal blood proteins (placental growth factor, soluble vascular endothelial growth factor receptor-1, pregnancy-associated plasma protein A). Such models require the transformation of biomarker data into multiples of the mean values by using population- and site-specific models. Previous studies have focused on a narrow window in gestation and have not included the maternal blood concentration of soluble endoglin, an important antiangiogenic factor up-regulated in preeclampsia. This study aimed (1) to develop models for the calculation of multiples of the mean values for mean arterial pressure and biochemical markers; (2) to build and assess the predictive models for preeclampsia based on maternal risk factors, the biophysical (mean arterial pressure) and biochemical (placental growth factor, soluble vascular endothelial growth factor receptor-1, and soluble endoglin) markers collected throughout pregnancy; and (3) to evaluate how prediction accuracy is affected by the presence of chronic hypertension and gestational age. This longitudinal case-cohort study included 1150 pregnant women: women without preeclampsia with (n=49) and without chronic hypertension (n=871) and those who developed preeclampsia (n=166) or superimposed preeclampsia (n=64). Mean arterial pressure and immunoassay-based maternal plasma placental growth factor, soluble vascular endothelial growth factor receptor-1, and soluble endoglin concentrations were available throughout pregnancy (median of 5 observations per patient). A prior-risk model for preeclampsia was established by using Poisson regression based on maternal characteristics and obstetrical history. Next, multiple regression was used to fit biophysical and biochemical marker data as a function of maternal characteristics by using data collected at 8 to 15+6, 16 to 19+6, 20 to 23+6, 24 to 27+6, 28 to 31+6, and 32 to 36+6 week intervals, and observed values were converted into multiples of the mean values. Then, multivariable prediction models for preeclampsia were fit based on the biomarker multiples of the mean data and prior-risk estimates. Separate models were derived for overall, preterm, and term preeclampsia, which were evaluated by receiver operating characteristic curves and sensitivity at fixed false-positive rates. (1) The inclusion of soluble endoglin in prediction models for all preeclampsia, together with the prior-risk estimates, mean arterial pressure, placental growth factor, and soluble vascular endothelial growth factor receptor-1, increased the sensitivity (at a fixed false-positive rate of 10%) for early prediction of superimposed preeclampsia, with the largest increase (from 44% to 54%) noted at 20 to 23+6 weeks (McNemar test, P<.05); (2) combined evidence from prior-risk estimates and biomarkers predicted preterm preeclampsia with a sensitivity (false-positive rate, 10%) of 55%, 48%, 62%, 72%, and 84% at 8 to 15+6, 16 to 19+6, 20 to 23+6, 24 to 27+6, and 28 to 31+6 week intervals, respectively; (3) the sensitivity for term preeclampsia (false-positive rate, 10%) was 36%, 36%, 41%, 43%, 39%, and 51% at 8 to 15+6, 16 to 19+6, 20 to 23+6, 24 to 27+6, 28 to 31+6, and 32 to 36+6 week intervals, respectively; (4) the detection rate for superimposed preeclampsia among women with chronic hypertension was similar to that in women without chronic hypertension, especially earlier in pregnancy, reaching at most 54% at 20 to 23+6 weeks (false-positive rate, 10%); and (5) prediction models performed comparably to the Fetal Medicine Foundation calculators when the same maternal risk factors and biomarkers (mean arterial pressure, placental growth factor, and soluble vascular endothelial growth factor receptor-1 multiples of the mean values) were used as input. We introduced prediction models for preeclampsia throughout pregnancy. These models can be useful to identify women at risk during the first trimester who could benefit from aspirin treatment or later in pregnancy to inform patient management. Relative to prediction performance at 8 to 15+6 weeks, there was a substantial improvement in the detection rate for preterm and term preeclampsia by using data collected after 20 and 32 weeks' gestation, respectively. The inclusion of plasma soluble endoglin improves the early prediction of superimposed preeclampsia, which may be valuable when Doppler velocimetry of the uterine arteries is not available.

Rheological analysis of sputum from patients with chronic bronchial diseases

Bronchial diseases are characterised by the weak efficiency of mucus transport through the lower airways, leading in some cases to the muco-obstruction of bronchi. It has been hypothesised that this loss of clearance results from alterations in the mucus rheology, which are reflected in sputum samples collected from patients, making sputum rheology a possible biophysical marker of these diseases and their evolution. However, previous rheological studies have focused on quasi-static viscoelastic (linear storage and loss moduli) properties only, which are not representative of the mucus mobilisation within the respiratory tract. In this paper, we extend this approach further, by analysing both quasi-static and some dynamic (flow point) properties, to assess their usability and relative performance in characterising several chronic bronchial diseases (asthma, chronic obstructive pulmonary disease, and cystic fibrosis) and distinguishing them from healthy subjects. We demonstrate that pathologies influence substantially the linear and flow properties. Linear moduli are weakly condition-specific and even though the corresponding ranges overlap, distinct levels can be identified. This directly relates to the specific mucus structure in each case. In contrast, the flow point is found to strongly increase in muco-obstructive diseases, which may reflect the complete failure of mucociliary clearance causing episodic obstructions. These results suggest that the analysis of quasi-static and dynamic regimes in sputum rheology is in fact useful as these regimes provide complementary markers of chronic bronchial diseases.